Table 1.
Overview of the clinical data.
Figure 1.
Distribution of immature and mature mDC, pDC, MΦ and CD163+ MΦ in cHL.
(A) CD1a staining (n = 101) with numerous small reactive tumor infiltrating DC besides few CD1a-negative HRS cells. (B) CD83 staining (n = 97) with small infiltrating DC besides CD83-positive HRS cells (arrow). (C) CD123 staining (n = 97) showing pDC with a plasmacytoid morphology besides CD123 negative HRS cells. (D) CD68 staining (n = 96) with a cytoplasmatic positivity of MΦ and (E) CD163 staining (n = 94) with a membranous and cytoplasmic positivity of TAM. Examples of cHL specimens are depicted all acquired with a Zeiss Imager.A1 (400x magnification, 40x/0.75 objective) using AxioVision SE64 Rel.4.8 software. (F) Box-Plots show the distribution of the different DC subtypes and MΦ in the investigated cHL cases. (G) Comparison of the distribution of DC and MΦ in EBV- and EBV+ cHL. Unpaired student's t-tests or Mann-Whitney-U-tests were conducted with *P<0.05, **P<0.01 and ***P<0.001. Data in (F) are indicated as Box-and-Whisker Plots with median. Data in (G) are indicated as mean with SEM.
Figure 2.
Influence of mature mDC and CD163+ MΦ on the clinical outcome of cHL patients.
Kaplan-Meier-Analysis of disease-specific survival (DSS) of cHL patients with regard to the numbers of infiltrating CD83+ mature mDC (A) and CD163+ MΦ (B, C). (A) The mean and (B) the 50th percentile and (C) the 75th percentile were used as cut-off, to define the groups with high and low numbers of CD83+ cell/mm2 and CD163+ cell/mm2.
Figure 3.
Expression of TNF, IL10 and TGFβ1 in cHL cases with high or low numbers of mature mDC and CD163+ MΦ.
RNA was isolated from FFPE cHL sections and subjected to qRT-PCR. The expression of TNF, IL10 and TGFβ1 was determined. cHL cases were grouped into subgroups with low or high numbers of CD83+ or CD163+ infiltrating cells using the 25. or 75. percentile as cut-off, respectively and compared to a control group of benign reactive lymphadenopathy (rLA). All values are shown as fold change compared to the measured cytokine expression in the L1236 cell line. Mann-Whitney-U-tests were conducted with *P<0.05, **P<0.01 and ***P<0.001 comparing either all cHL cases with rLA or cHL cases with presumably adverse prognosis (CD83 low and CD163 high) with those with an expected better prognosis (CD83 high and CD163 low). Single bars represent the mean + SD of triplicates of single analyzed cases, each.
Figure 4.
Expression of GM-CSF and M-CSF in cHL cases with high or low numbers of mature mDC and CD163+ MΦ.
RNA was isolated from FFPE cHL sections and subjected to qRT-PCR. The expression of GM-CSF and M-CSF was determined. cHL cases were grouped into subgroups with low or high numbers of CD83+ or CD163+ infiltrating cells using the 25. or 75. percentile as cut-off, respectively and compared to a control group of benign reactive lymphadenopathy (rLA). All values are shown as fold change compared to the measured cytokine expression in the L1236 cell line. Mann-Whitney-U-tests were conducted with *P<0.05, **P<0.01 and ***P<0.001 comparing either all cHL cases with rLA or cHL cases with presumably adverse prognosis (CD83 low and CD163 high) with those with an expected better prognosis (CD83 high and CD163 low). Single bars represent the mean + SD of triplicates of single analyzed cases, each.
Figure 5.
Expression of IL4 and IL13 in cHL cases with high or low numbers of mature mDC and CD163+ MΦ.
RNA was isolated from FFPE cHL sections and subjected to qRT-PCR. The expression of IL4 and IL13 was determined. cHL cases were grouped into subgroups with low or high numbers of CD83+ or CD163+ infiltrating cells using the 25. or 75. percentile as cut-off, respectively and compared to a control group of benign reactive lymphadenopathy (rLA). All values are shown as fold change compared to the measured cytokine expression in the L1236 cell line. Mann-Whitney-U-tests were conducted with *P<0.05, **P<0.01 and ***P<0.001 comparing either all cHL cases with rLA or cHL cases with presumably adverse prognosis (CD83 low and CD163 high) with those with an expected better prognosis (CD83 high and CD163 low). Single bars represent the mean + SD of triplicates of single analyzed cases, each.
Figure 6.
Characterization of the maturation and activation of moDC.
(A) moDC were treated for 24 h with TNFα, IL10 and TGFβ (each 10 ng/ml), 50% L1236 or HDLM2 supernatants (SN) or with (B) 50% L1236 supernatants and either an IgG1 isotype control, an neutralizing TNFα antibody or a therapeutically applied TNFα antibody (infliximab). Supernatants were generated at a concentration of 1×106 cells/ml medium after 48 h incubation. The expression of the DC activation marker CD40, the monocyte marker CD14 and the DC maturation marker CD83 was determined by flow cytometry. Mean fluorescence intensity (MFI) is indicated on the bar charts. Paired student's t-tests or Wilcoxon signed-rank tests were conducted with *P<0.05, **P<0.01 and ***P<0.001.
Figure 7.
Characterization of the polarization profile of monocytes, MΦGM-CSF (MΦ-1 model) and MΦM-CSF (MΦ-2 model).
Monocytes were cultured for 5 days in complete medium or 50% L1236 or HDLM2 SN. MΦGM-CSF and MΦM-CSF were treated for 24 h with medium or 50% L1236 or HDLM2 SN. The expression of different MΦ lineage markers was determined, including CD68, CD163, CD206 and HLA-DR. Mean fluorescence intensity (MFI) is indicated on the bar charts. Paired student's t-tests or Wilcoxon signed-rank tests were conducted with *P<0.05, **P<0.01 and ***P<0.001.
Figure 8.
Effects of moDC, MΦGM-CSF and MΦM-CSF on the proliferation of L1236 cells.
(A) L1236 cells were incubated with 90% moDC-, MΦGM-CSF - or MΦM-CSF - supernatants in the presence of CFSE for 96 h. Supernatants were generated at a concentration of 5×105 cells/ml incubated for 48 h. Mean fluorescence intensity (MFI) is indicated on the bar charts as determined by flow cytometry. (B) L1236 cells were incubated with MΦGM-CSF - or MΦM-CSF -supernatants or (B and C) co-cultured at a ratio of 6∶1 in the presence of BrdU for 48 h. BrdU was stained using a BrdU-specific primary antibody and an Alexa Fluor 488-coupled secondary antibody. Fluorescence intensity (FI) was measured by a fluorescence reader. All micrographs in (C) were acquired with an Olympus IX81 (100x magnification, 10x/0.30 Ph1 objective) using cellSens dimension software. Paired student's t-tests, Wilcoxon signed-rank tests or Mann-Whitney-U-tests were conducted with *P<0.05, **P<0.01 and ***P<0.001. Data are indicated as mean with SEM of 6-9 (A) or 6-8 (B) independent experiments.